A microplate-based facultative anaerobic microbial culture method and its application

Abstract

The present invention belongs to the technical field of microbial culture, and discloses a method and application of facultative anaerobic microbial culture based on a microplate. Method: 1) Gellan gum is mixed with a culture medium, melted, and a suspension culture medium is obtained; the expanded cultured facultative anaerobic microorganisms are diluted with the suspension culture medium to obtain an indicator bacteria suspension; 2) A phosphate solution containing glucose is added to the outermost circle wells of the microplate, an indicator bacteria suspension is added to the inner wells, a glucose oxidase solution is added to the outermost circle wells, the plate is sealed, and the plate is statically cultured. The present invention solves the problem of reading errors caused by edge effects and bacterial sedimentation in traditional microplate culture, significantly improves data reliability, and reduces costs and operational complexity. The method of the present invention is used to detect the activity of antibacterial substances in facultative anaerobic bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial cultivation, and in particular relates to a microplate-based facultative anaerobic microbial cultivation method and application. Background Art

[0002] The ease of manipulation and metabolic diversity of facultative anaerobic microorganisms as model organisms make them indispensable tools for antibacterial activity testing. A variety of assay methods can be used to efficiently and accurately assess the antibacterial effects of compounds, providing important evidence for the development and clinical application of antibacterial drugs. Microplates are used as culture vessels, and turbidimetric analysis is used to measure the change in absorbance of each well before and after incubation of facultative anaerobic microorganisms, enabling high-throughput testing.

[0003] Due to the structure of microplates and the characteristics of turbidimetric detection, the following problems exist in the process of culturing facultative anaerobic microorganisms using microplates: (1) Bacterial sedimentation: Microorganisms tend to settle to the bottom of the wells during static culture, resulting in low absorbance (OD) values ​​or unstable readings. This error is exacerbated, especially during long-term culture or high-density culture. (2) Edge effect: Due to differences in temperature, humidity, and evaporation rates, the growth rate of microorganisms in the peripheral wells of traditional microplates is inconsistent with that in the central wells, resulting in significant deviations in the test data (such as OD values ​​and fluorescence intensity). (3) Insufficient control of facultative anaerobic conditions: Conventional microplates make it difficult to accurately control oxygen concentration, which affects the metabolic state of facultative anaerobic microorganisms.

[0004] Research on methods for cultivating facultative anaerobic microorganisms mainly focuses on the modification of cultivation devices, such as: Chinese utility model with authorization publication number CN218755761U, titled: A facultative anaerobic microorganism cultivation device; Chinese utility model with authorization publication number CN218058988U, titled: Cultivation device for facultative anaerobic antimony oxidizing bacteria and strictly anaerobic antimony oxidizing bacteria. No relevant literature on methods for cultivating facultative anaerobic microorganisms has been found.

[0005] Research on microplate-based detection of antibacterial substances has primarily focused on: 1. Increasing the number of single tests, i.e., high-throughput testing: Designing high-throughput turbidimetric detection methods, such as large-scale screening techniques based on 96-well or 384-well plates, can simultaneously test a large number of samples for antibacterial activity, accelerating the screening of antibacterial substances. For example, Chinese invention publication CN117737185A discloses a high-throughput screening method for antibacterial activity, comprising taking a culture plate, setting up a negative control group, a positive control group, and a drug experimental group for the target strain in the culture plate to obtain a sample plate; culturing the strain on the sample plate to a preset concentration of the target strain; and testing the antibacterial activity of the cultured sample plate, thereby achieving high-throughput testing. 2. Combining with other methods can shorten detection time and improve detection sensitivity and accuracy. For example, the Chinese invention patent application number CN201110127094.5 discloses a method for determining the minimum inhibitory concentration of a drug. This invention uses two high-quality fluorescent dyes and uses fluorescence to determine the minimum inhibitory concentration of a drug against enterococci, successfully shortening the detection time to 4 hours. This solves the problems of the traditional broth dilution method with long culture time and human factors in the visual interpretation of easily precipitating bacteria.

[0006] Bacterial sedimentation mostly relies on oscillation culture, but it may interfere with the facultative anaerobic environment and increase the complexity of the equipment. Furthermore, due to the small volume of a single hole in the microplate, it cannot be shaken more vigorously, so oscillation culture is not applicable. In order to make the measurement results more accurate, some experimenters will use a pipette to mix the liquid to be tested before testing, but this method is cumbersome and prone to bubbles. Due to the scattering and refraction of bubbles, the overall optical uniformity of the liquid to be tested is changed, making the measurement of the absorbance value unstable and inaccurate. In addition, the position and number of bubbles in the liquid are often uncertain, and the state of the bubbles may be different each time they are measured. This will result in differences in the absorbance values ​​obtained for each measurement, reducing the reproducibility and accuracy of the measurement. The edge effect is currently often solved by abandoning the peripheral holes or data correction, but this method only solves the "edge effect" caused by heat transfer and cannot solve the impact of differences in oxygen concentration.

[0007] In summary, existing studies have not considered the uneven distribution of bacteria in static culture microplates and the detection errors and poor repeatability caused by the "edge effect", nor have they considered the different growth conditions of facultative anaerobic microorganisms under different oxygen concentrations.

[0008] Therefore, it is necessary to develop a microplate-based method for culturing facultative anaerobic microorganisms. This method, used to detect the activity of antibacterial substances in facultative anaerobic bacteria, can eliminate both measurement errors caused by uneven bacterial distribution and uneven growth due to the "edge effect." This method is simple to operate, provides accurate results, and is suitable for high-throughput culture. Summary of the Invention

[0009] To address the issues of uneven bacterial distribution and the "edge effect" that can lead to detection errors and poor reproducibility in static culture microplates, and to develop a method that is simple to operate, provides accurate results, and enables high-throughput screening of antibacterial substances, the present invention provides a microplate-based method for culturing facultative anaerobic microorganisms and its application. This method involves adding a glucose oxidase solution and a glucose solution to the outermost culture wells (i.e., the edge wells) of the microplate. The oxygen concentration within the plate is controlled by adjusting the glucose concentration. A suspension medium containing gellan gum is then diluted with the bacterial solution to obtain a bacterial suspension. This suspension is then added to the inner wells and statically cultured. This method ensures the stable suspension and growth of sensitive indicator bacteria in the culture medium, eliminating the "edge effect" that affects the growth of facultative anaerobic microorganisms. It also eliminates detection errors caused by bacterial sedimentation at the bottom of the container during liquid culture methods, achieving high-throughput and accurate detection of facultative anaerobic microbial growth. The method is useful for detecting the activity of antibacterial substances in facultative anaerobic bacteria.

[0010] The purpose of the present invention is achieved through the following technical solutions.

[0011] A microplate-based method for culturing facultative anaerobic microorganisms comprises the following steps:

[0012] 1) Mixing gellan gum and a culture medium for facultative anaerobic microorganisms, melting them, and obtaining a suspension culture medium; diluting the expanded cultured facultative anaerobic microorganisms with the suspension culture medium to obtain an indicator bacteria suspension;

[0013] 2) Add a phosphate solution containing glucose to the outermost wells of the microplate, add an indicator bacteria suspension to the inner wells of the microplate, then add a glucose oxidase solution to the outermost wells of the microplate, and seal the microplate.

[0014] 3) Static culture.

[0015] The gellan gum in step 1) is deacylated gellan gum or low-acyl gellan gum, and the content of the gellan gum in the suspension culture medium is 0.2 g / L to 0.4 g / L.

[0016] The facultative anaerobic microorganism is Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, Enterococcus faecalis, Streptococcus thermophilus, Listeria, yeast or Candida.

[0017] The viable count of the indicator bacteria suspension is 10 4 ~10 7 CFU / mL; that is, the expanded culture of facultative anaerobic microorganisms is diluted with the suspension culture medium so that the viable count after dilution is 10 4 ~10 7 CFU / mL.

[0018] The glucose concentration in the glucose-containing phosphoric acid solution in step 2) is 50 g / L to 300 g / L. The glucose-containing phosphoric acid solution is obtained by dissolving glucose in a phosphate buffer solution having a pH of 4.0 to 7.0 and a concentration of 0.02 mol / L to 0.05 mol / L.

[0019] The concentration of the glucose oxidase solution is 0.5 g / L to 50 g / L. The glucose oxidase solution is obtained by dissolving glucose oxidase in a phosphate buffer solution with a concentration of 0.02 mol / L to 0.05 mol / L and a pH of 5.0 to 7.0.

[0020] The volume ratio of the glucose-containing phosphate solution to the glucose oxidase solution is 50-100:50-100, preferably 1:1. The amount of the glucose-containing phosphate solution added to the culture well is 50-100 μL, and the amount of the indicator bacteria suspension added to the culture well is 50-150 μL.

[0021] The static culture conditions in step 3) are placing the cells in an incubator at 28-45° C. for 4-72 hours.

[0022] The melting temperature in step 1) is 90-100°C. The suspension culture medium in step 1) specifically refers to mixing gellan gum with a culture medium for facultative anaerobic microorganisms, heating with stirring at 90-100°C, melting until transparent and clear, sterilizing, and cooling. Sterilization conditions: Sterilize at 115-121°C for 15-30 minutes.

[0023] The expanded culture of facultative anaerobic microorganisms refers to inoculating the strains of facultative anaerobic microorganisms into a sterilized culture medium, culturing at 28-45°C for 6-16 hours to obtain an activated bacterial solution; taking the activated bacterial solution and inoculating it again into a fresh culture medium, and expanding the culture at 28-45°C.

[0024] The expanded culture is static culture or shaker culture (rotation speed is 1-250 rpm) for 8-24 h.

[0025] The method of the invention is used for detecting the activity of antibacterial substances of facultative anaerobic bacteria, and comprises high-throughput screening of antibacterial substances and determination of minimum antibacterial concentration.

[0026] A method for detecting the activity of an antibacterial substance of facultative anaerobic bacteria comprises the following steps:

[0027] S1. Mixing gellan gum and a culture medium of facultative anaerobic microorganisms, and melting them to obtain a suspension culture medium;

[0028] S2. diluting the expanded cultured facultative anaerobic microorganisms with the suspension culture medium to obtain an indicator bacteria suspension;

[0029] S3, after the antibacterial substance is prepared into a mother solution, it is diluted with a suspension culture medium to obtain an antibacterial substance suspension;

[0030] S4. Divide the inner wells of the microplate except the outermost circle of wells into a test area, a bacterial liquid growth control area, and a negative control area; add a phosphate solution containing glucose to the outermost circle of wells of the microplate, add a suspension culture medium to the other inner wells, then add an antibacterial substance suspension to the wells in the test area, then add an indicator bacteria suspension to the wells in the test area and the wells in the bacterial liquid growth control area, add a suspension culture medium to the wells in the negative control area, and finally add a glucose oxidase solution to the outermost circle of culture wells, and seal the microplate;

[0031] S5, static culture;

[0032] S6. Measure the absorbance of each well at 550-600 nm.

[0033] In step S4, the antibacterial substance suspension is diluted with a suspension culture medium to form different concentrations in the wells of the test area.

[0034] Specifically, the suspension of the antibacterial substance in the wells of the test area is gradiently diluted with the suspension culture medium, so that the antibacterial substance forms a concentration gradient in the wells.

[0035] The gradient dilution is specifically performed using a two-fold gradient dilution method.

[0036] The wells in the test area are divided into area one and area two. The antibacterial substance suspension in the wells of area one is gradiently diluted with a suspension culture medium so that the antibacterial substance forms a concentration gradient; the wells in area two are wells for parallel tests, i.e., parallel wells; each well in area one corresponds to a concentration, and each concentration is provided with at least one parallel well in area two (e.g., 1 to 4 parallel wells).

[0037] Specifically, the antibacterial substance suspension in one row of wells in the test area is gradiently diluted with suspending culture medium, such that a concentration gradient of the antibacterial substance is formed within the wells; another row, two rows, three rows, or four rows of wells are parallel wells, and the concentration of the antibacterial substance in the parallel wells is the same as described above for the concentration gradient. For example, the antibacterial substance suspension in the first row of wells is gradiently diluted with suspending culture medium, such that a concentration gradient of the antibacterial substance is formed within the wells; the concentration of the antibacterial substance in wells 1 through 4 of the second, third, fourth, and fifth rows is the same as that in the first row.

[0038] In step S6, a curve is drawn based on the absorbance value, with the concentration of the antibacterial substance as the abscissa and the OD600 value as the ordinate, and the inflection point is the minimum inhibitory concentration.

[0039] The microplate in step S4 is a 96-well plate, a 48-well plate, a 24-well plate, a 384-well plate, or a 1536-well plate.

[0040] The concentration of the antibacterial substance suspension in step S3 is 200-1000 μg / mL.

[0041] The conditions in steps S1 and S2 are the same as those defined in the above-mentioned method for culturing facultative anaerobic microorganisms.

[0042] The glucose-containing phosphoric acid solution and glucose oxidase solution in step S4 are the same as those defined in the above-mentioned method for culturing facultative anaerobic microorganisms.

[0043] The static culture condition in step S5 is to place the culture in an incubator at 28-45° C. for 4-72 hours.

[0044] The present invention adds gellan gum to a liquid culture medium. After heating and hydration, the gellan gum forms a double helix structure, which further aggregates and cross-links through interactions such as hydrogen bonds and van der Waals forces, forming a three-dimensional network. Suspended macromolecular particles (such as macromolecular nutrients and microorganisms) are captured in this three-dimensional network and confined within its pores, preventing them from settling or aggregating freely, thereby achieving an excellent suspension effect. This network structure can withstand certain external forces, maintaining the stability of the system and preventing particles from sinking due to gravity, thereby achieving suspended microbial culture and eliminating detection errors caused by uneven cell distribution. Furthermore, by adjusting the concentration of gellan gum, the liquid maintains a certain degree of fluidity while ensuring particle suspension, allowing small molecules such as water to flow freely within the gaps in the three-dimensional network. The present invention adds a phosphoric acid solution containing glucose oxidase and glucose to the outermost circle of the microplate, utilizes glucose oxidase to catalyze the reaction in which glucose combines with oxygen to generate gluconic acid, and regulates the concentration of oxygen by adjusting the concentration of glucose, thereby regulating the growth of facultative anaerobic bacteria; at the same time, the outermost circle of holes is filled with the solution, which can ensure the uniformity of the temperature of the internal holes, ultimately achieving the purpose of eliminating the "edge effect" of the microplate and regulating the oxygen concentration in the microplate.

[0045] Compared with the prior art, the present invention has the following advantages and effects:

[0046] The present invention utilizes the characteristic of gellan gum forming a three-dimensional network at low concentrations to prepare a suspension culture medium. Compared with biopolysaccharides such as agar and carrageenan, gellan gum has the advantages of antimicrobial utilization, high gel transparency, good thermal stability, and a wide applicable pH range. The suspension culture medium prepared by the present invention is different from general agar gel. It has a certain fluidity at room temperature, which is convenient for experimental operation. The three-dimensional network structure formed by gellan gum can suspend macromolecules such as microbial cells and proteins to prevent them from settling; for small molecules such as water, it does not restrict their movement. It enables microorganisms to grow uniformly in the culture medium, avoids the occurrence of sedimentation and stratification, and also maintains the uniformity of light transmittance of the sample. In addition, since the growth of facultative anaerobic microorganisms is affected by oxygen concentration and "edge effect", the present invention arranges glucose oxidase and glucose solution in the outermost circle of the microplate, consumes oxygen in the outer holes through catalytic reaction and balances the evaporation rate, reduces the edge effect, and thus maintains the consistency of the growth conditions of facultative anaerobic microorganisms.

[0047] The present invention does not require special instruments and reagents, but uses common and affordable reagents to achieve suspension culture of facultative anaerobic bacteria in liquid culture medium. At the same time, it eliminates the "edge effect" and the influence of oxygen concentration on the growth of facultative anaerobic indicator bacteria, and solves the problems of complex operation, small single detection volume, and large error in results of traditional microplate culture methods. It is suitable for high-throughput detection scenarios, significantly improves data reliability, and reduces costs and operational complexity. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a schematic diagram of microplate sample loading in Example 1;

[0049] Figure 2 The positions of the holes in the microplate in Examples 1-2 and Comparative Examples 1-3;

[0050] Figure 3 is the pore distribution of each group in Example 3 and Comparative Example 4;

[0051] Figure 4 This is a schematic diagram of the two-fold gradient dilution in Example 3. DETAILED DESCRIPTION

[0052] The present invention will be described in further detail below with reference to specific examples, but the embodiments of the present invention are not limited thereto.

[0053] Example 1: Static suspension culture of Escherichia coli in 96-well plates

[0054] Step 1: Prepare 500 mL of LB liquid medium according to the culture requirements for E. coli. The medium composition is as follows: 5.0 g tryptone, 2.5 g yeast extract, and 2.5 g sodium chloride. Dissolve in water, adjust the pH to 7.0 ± 0.2, and make up to 500 mL.

[0055] Weigh 0.20 g of deacylated gellan gum (gellan gum addition amount is 0.4 g / L) and add it to the above culture medium. After uniform dispersion, heat and melt at 90-100°C until transparent and clear. Dispense into test tubes and Erlenmeyer flasks, sterilize at 121°C for 20 min, and cool to obtain a suspension culture medium.

[0056] Step 2: Take 50 μL of E. coli stored in the glycerol tube, inoculate it into a 10 mL sterilized ordinary LB medium test tube, and culture it in a shaker at 37°C and 180 rpm for 12-16 hours to obtain an activated bacterial solution; take 500 μL of the activated strain and inoculate it again into 100 mL of fresh ordinary LB medium, and expand the culture in a shaker at 37°C and 180 rpm for 12 hours to obtain an E. coli suspension; dilute the activated E. coli suspension with the suspension medium prepared in step 1 so that the number of viable bacteria after dilution is 1×10 4 CFU / mL, that is, the bacterial suspension to be tested;

[0057] Step 3: Take a 96-well plate and add 100 μL of 300 g / L glucose phosphate solution (solvent is 0.05 mol / L, pH 5.0 PBS solution) to the outermost well of the culture plate. Add the diluted test bacterial suspension to the other positions of the 96-well plate (the inner wells are the test wells); then add 100 μL of 50 g / L glucose oxidase phosphate solution (solvent is 0.05 mol / L, pH 5.0 PBS solution) to the outermost well of the culture plate; finally, cover the lid and apply sealing tape to the outer ring of the microplate to seal;

[0058] Step 4: Place the sample-loaded microplate in a 37°C incubator and use a microplate reader to measure the absorbance of each well at 600 nm at 0 h, 4 h, 24 h, 48 h, and 72 h.

[0059] The schematic diagram of microplate loading in this embodiment is as follows Figure 1 As shown, the outermost well of the culture plate is filled with glucose oxidase solution, and the other wells are test wells, where the bacterial suspension to be tested is added. Figure 1 The black circles in the middle are the wells where the glucose oxidase solution is added, and the white circles are the test wells where the bacterial suspension to be tested is added.

[0060] The positions of the holes in the microplate of this embodiment are as follows Figure 2As shown, the microporous plate includes peripheral holes (i.e., outermost circle holes), inner circle holes 1, inner circle holes 2, and a central hole.

[0061] The OD600 value of each well at 4h, 24h, 48h and 72h was calculated, and the results are shown in Tables 1 to 4. Compared with Comparative Example 1 (adding phosphoric acid aqueous solution to the outermost circle of the 96-well plate and using ordinary LB culture medium) and Comparative Example 2 (using ordinary LB culture medium and inoculating E. coli in all 96 wells), the absorbance values ​​of the samples after culturing E. coli for 4h, 24h, 48h and 72h in Example 1 were more stable than those of the other two, and the variance and coefficient of variation were smaller than those of the comparative examples, indicating that in Example 1, the detection data of the absorbance value of E. coli was more stable and had a smaller error. Comparing the samples at different positions of the 96-well plate, the data deviation between the center hole and the inner circle holes in Example 1 was small (the position of each well in the microplate in this embodiment is as shown in the figure). Figure 2 As shown), the data deviations of Comparative Examples 1 and 2 are large (coefficient of variation CV>5%), indicating that the OD600 data is affected by the position of the well plate, and Example 1 of the present invention can eliminate this influence.

[0062] Table 1 Absorbance values ​​(△OD600) of E. coli samples after 4 h of culture

[0063] △OD600= OD600 (4h) - OD600 (0h)

[0064]

[0065] Table 2 Absorbance values ​​(△OD600) of E. coli samples after 24 h of culture

[0066] △OD600= OD600 (24h)-OD600 (0h)

[0067]

[0068] Table 3 Absorbance values ​​(△OD600) of E. coli samples after 48 h of culture

[0069] △OD600= OD600 (48h)-OD600 (0h)

[0070]

[0071] Table 4 Absorbance values ​​(OD600) of E. coli samples after 72 h of culture

[0072] △OD600= OD600 (72h)-OD600 (0h)

[0073]

[0074] Example 2: Static suspension culture of Staphylococcus aureus in 96-well plates

[0075] Step 1: Prepare 1000 mL of nutrient broth (NB) according to the culture requirements for Staphylococcus aureus. The composition of the NB medium is as follows: peptone: 10 g (w / v), beef extract powder: 3.0 g (w / v), sodium chloride: 5.0 g (w / v). Dissolve in water, adjust the pH to 7.4 ± 0.2, and make up to 1000 mL.

[0076] Weigh 0.10 g of deacylated gellan gum (gellan gum addition amount is 0.2 g / L) and add it to the above culture medium. After uniform dispersion, heat and melt at 90-100°C until transparent and clear. Dispense into test tubes and Erlenmeyer flasks, sterilize at 121°C for 20 min, and cool to obtain a suspension culture medium.

[0077] Step 2: Take 50 μL of Staphylococcus aureus stored in a glycerol tube and inoculate it into a 10 mL sterilized ordinary NB culture medium test tube. Incubate it in a shaker at 37°C and 150 rpm for 12-16 h to obtain an activated bacterial solution. Take 500 μL of the activated strain and inoculate it again into 100 mL of fresh ordinary NB culture medium. Continue incubating it in a shaker at 37°C and 150 rpm for 8 hours to obtain a Staphylococcus aureus suspension. Dilute the activated Staphylococcus aureus suspension with the suspension culture medium so that the viable count after dilution is 1×10 5 CFU / mL, that is, the bacterial suspension to be tested;

[0078] Step 3: Take a 96-well plate, empty the outermost circle of the culture plate, and add 100 μL of 100 g / L glucose phosphate solution (the solvent is 0.04 mol / L, pH 5.5 phosphate solution) to the outer wells; add the diluted test bacterial suspension to the other positions of the 96-well plate (inner circle well 1, inner circle well 2, center well) (the position of each well is as shown in the figure). Figure 2 ), then add 100 μL of 1.0 g / L glucose oxidase phosphate solution (solvent is 0.04 mol / L phosphate solution, pH 5.5) to the outermost circle wells, cover the lid and apply sealing tape to the outer circle of the microplate to seal it; the positions of the wells in this embodiment are as shown Figure 2 As shown;

[0079] Step 4: Place the sample plate in a 37°C incubator and use a microplate reader to measure the absorbance of each well at 600 nm at 0 h and 48 h.

[0080] The positions of the holes in the microplate in this embodiment are as follows: Figure 2 As shown, the microporous plate includes peripheral holes, inner circle holes 1, inner circle holes 2, and a central hole.

[0081] The results, shown in Table 5, show that compared to Comparative Example 3 (using standard NB medium and inoculating S. aureus in all 96 wells), the absorbance values ​​of the S. aureus samples in Example 2 after 48 hours of culture were slightly higher, while the variance and coefficient of variation were significantly lower than those in Comparative Example 3. This indicates that Example 2 exhibited better growth of S. aureus and greater data stability with reduced error. Comparing absorbance values ​​at different locations within the plate revealed good uniformity across the wells in Example 2. However, the absorbance values ​​at different locations in Comparative Example 3 varied significantly, with a coefficient of variation of 16.73%, exceeding 5%, indicating a high degree of data dispersion.

[0082] Table 5 Absorbance values ​​of samples after 48h of Staphylococcus aureus culture

[0083] △OD600= OD600 (48h)-OD600 (0h)

[0084]

[0085] Example 3: Determination of the Minimum Inhibitory Concentration of Nisin against Enterococcus faecalis

[0086] Step 1: Prepare 500 mL of LB liquid medium according to the culture requirements for Enterococcus faecalis. The composition of the medium, by mass to volume ratio, is as follows: tryptone: 5.0 g (w / v), yeast powder: 2.5 g (w / v), sodium chloride: 2.5 g (w / v). Dissolve in water, adjust the pH to 7.0 ± 0.2, and make up to 500 mL.

[0087] Weigh 0.15 g of deacylated gellan gum (gellan gum addition amount is 0.3 g / L) and add it to the above culture medium. Heat at 90-100°C to melt until transparent and clear. Aliquot into test tubes and Erlenmeyer flasks, sterilize at 115°C for 30 min, and cool to obtain a suspension culture medium.

[0088] Step 2: Weigh 0.2000 g of Nisin (nisin) powder and dissolve it in 100 mL of sterile triple-distilled water to prepare a 2000 μg / mL Nisin stock solution. Sterilize the solution by filtration through a sterile 0.22 μm fiber filter in a laminar flow hood, aliquot, and store at -20°C. Thaw the solution before experimentation and dilute it to 400 μg / mL using the suspension medium prepared in Step 1.

[0089] Step 3: Take 50 μL of Enterococcus faecalis stored in the glycerol tube and inoculate it into a 10 mL sterilized ordinary LB medium test tube, and culture it in a shaker at 37°C and 180 rpm for 6-8 h to obtain an activated bacterial solution; take 500 μL of the activated strain and inoculate it again into 100 mL of fresh ordinary LB medium, and expand the culture under the conditions of a shaker at 37°C and 180 rpm to obtain an Enterococcus faecalis suspension; dilute the activated Enterococcus faecalis suspension with the suspension medium in step 1 so that the number of viable bacteria after dilution is 1×10 7 CFU / mL, which is the indicator bacteria suspension to be tested;

[0090] Step 4: Take a 96-well plate and add 100 μL of 50 g / L glucose phosphate solution (solvent is 0.02 mol / L, pH 7.0 PBS solution) to the outermost circle of wells. Add 100 μL of suspension culture medium to the other wells (test wells, negative control wells, and bacterial growth control wells). The well distribution of each group in this example is as follows: Figure 3 As shown, B2~B11, C2~C11, D2~D11, E2~E11 are experimental groups, F2~F11 are set as bacterial growth control groups, and G2~G11 are set as negative control groups;

[0091] Step 5: Take 100 μL of 400 μg / mL Nisin from step 2 and add it to well B2. Use the two-fold gradient dilution method (i.e., each dilution step reduces the concentration of the previous step solution to 1 / 2 of the original, thus forming a series of isocratic dilution concentration gradients). Use the suspension culture medium to perform gradient dilutions in wells B3 to B11 (see Figure 4 ), the Nisin concentrations in wells B2 to B11 are shown in Table 6; 4 parallel runs are performed for each sample, i.e., the operations for wells C2 to C11, D2 to D11, and E2 to E11 are the same as those for wells B2 to B11; the schematic diagram of the two-fold gradient dilution is shown in Figure 4 As shown;

[0092] Step 6: In a 96-well plate, add 100 μL of Enterococcus faecalis suspension to the growth control and experimental groups in the inner wells, such as wells numbered F2-F11 as the bacterial growth control, and wells numbered G2-G11 as the negative control. Add 100 μL of sterile suspension culture medium to the negative control. Finally, add 100 μL of 0.5 g / L glucose oxidase phosphate solution (solvent: 0.02 mol / L, pH 7.0 phosphate buffer solution) to the outer wells and seal with sealing tape.

[0093] Step 7: Place the sample plate in a 37°C incubator for incubation. According to the OD value determination method, use a microplate reader to read and record the OD600 value of each well of the 96-well plate before and after 24 hours of incubation. Calculate the difference in OD600 values ​​before and after drug action. The drug concentration in the well with an OD value change of less than 0.05 is the minimum inhibitory concentration (MIC).

[0094] The pore distribution of each group in this embodiment is as follows Figure 3 The glucose oxidase phosphate solution is added to the outermost circle of wells in the microplate last. The outermost circle of wells is recorded as the glucose oxidase solution wells. The wells in the microplate are also divided into test wells, negative control wells, and bacterial growth control wells.

[0095] Figure 4 This is a schematic diagram of the two-fold gradient dilution in Example 3.

[0096] The results, shown in Table 6, show that the minimum inhibitory concentration of nisin in Example 3 and Comparative Example 4 was consistent, both at 6.25 μg / mL. The absorbance CV values ​​for the examples were lower than those for the comparative examples, demonstrating good data stability and low deviation. When the concentration of nisin was lower than the MIC, the absorbance values ​​for the examples were higher than those for the comparative examples, indicating that the growth of Staphylococcus aureus in the examples was better than that in the comparative examples.

[0097] Table 6 Comparison of data before and after 24h culture of Example 3 and Comparative Example 4

[0098]

[0099] In the table: △OD600 = OD600 (24 h) - OD600 (0 h)

[0100] Note: CV is the coefficient of variation, that is, CV = standard deviation / mean * 100%, which is used to reflect the stability of the data. CV ≥ 5% is considered unstable.

[0101] Comparative Example 1: Static Culture of Escherichia coli in 96-Well Plates (Method 1)

[0102] Step 1: Prepare 500 mL of LB liquid medium according to the culture requirements for E. coli. The medium composition is as follows: tryptone: 5.0 g (w / v), yeast powder: 2.5 g (w / v), sodium chloride: 2.5 g (w / v). Dissolve in water, adjust the pH to 7.0 ± 0.2, and make up to 500 mL.

[0103] Dissolve the above culture medium and dispense it into test tubes and Erlenmeyer flasks, sterilize at 121°C for 20 min, and cool to obtain a suspension culture medium;

[0104] Step 2: Take 50 μL of E. coli stored in the glycerol tube and inoculate it into a 10 mL sterilized ordinary LB medium test tube, place it in a shaker at 37°C and 180 rpm for 12-16 hours to obtain an activated bacterial solution; take 500 μL of the activated strain and inoculate it again into 100 mL of fresh ordinary LB medium, and expand the culture at 37°C and 180 rpm for 12 hours; dilute the activated E. coli suspension with the suspension medium prepared in step 1 so that the number of viable bacteria after dilution is 1×10 4 CFU / mL, that is, the bacterial suspension to be tested;

[0105] Step 3: Take a 96-well plate, empty the outermost circle (peripheral wells) of the culture plate, and add 200 μL of phosphoric acid solution (0.05 mol / L, pH 5.0); add the diluted test bacterial suspension to the other positions of the 96-well plate (inner circle well 1, inner circle well 2, and the center well), cover the lid, and apply sealing tape to the outer circle of the microplate to seal;

[0106] Step 4: Place the sample plate in a 37°C incubator and use a microplate reader to measure the absorbance of each well at 600 nm at 0 h, 24 h, 48 h, and 72 h.

[0107] The positions of the holes in the microplate in this comparative example are as follows: Figure 2 As shown, the microporous plate includes peripheral holes, inner circle holes 1, inner circle holes 2, and a central hole.

[0108] Comparative Example 2: Static Culture of Escherichia coli in 96-well Plates (Method 2)

[0109] Step 1: Prepare 500 mL of LB liquid medium according to the culture requirements for E. coli. The medium composition is as follows: tryptone: 5.0 g (w / v), yeast powder: 2.5 g (w / v), sodium chloride: 2.5 g (w / v). Dissolve in water, adjust the pH to 7.0 ± 0.2, and make up to 500 mL.

[0110] Dissolve the above culture medium and dispense it into test tubes and Erlenmeyer flasks, sterilize at 121°C for 20 min, and cool to obtain a suspension culture medium;

[0111] Step 2: Take 50 μL of E. coli stored in the glycerol tube and inoculate it into a 10 mL sterilized ordinary LB medium test tube, place it in a shaker at 37°C and 180 rpm for 12-16 hours to obtain an activated bacterial solution; take 500 μL of the activated strain and inoculate it again into 100 mL of fresh ordinary LB medium, and expand the culture at 37°C and 180 rpm for 12 hours; dilute the activated E. coli suspension with the suspension medium prepared in step 1 so that the number of viable bacteria after dilution is 1×104 CFU / mL, that is, the bacterial suspension to be tested;

[0112] Step 3: Take a 96-well plate and add the diluted test bacterial suspension to all wells (outer wells, inner circle wells, and center wells). Cover the plate with a lid and seal the outer ring of the plate with sealing tape.

[0113] Step 4: Place the sample plate in a 37°C incubator and use a microplate reader to measure the absorbance of each well at 600 nm at 0 h, 24 h, 48 h, and 72 h.

[0114] The positions of the holes in the microplate in this comparative example are as follows: Figure 2 As shown, the microporous plate includes peripheral holes, inner circle holes 1, inner circle holes 2, and a central hole.

[0115] Comparative Example 3: Static culture of Staphylococcus aureus in 96-well plates

[0116] Step 1: Prepare 1000 mL of nutrient broth (NB) according to the culture requirements for Staphylococcus aureus. The composition of the NB medium is as follows: peptone: 10 g (w / v), beef extract powder: 3.0 g (w / v), sodium chloride: 5.0 g (w / v). Dissolve in water, adjust the pH to 7.4 ± 0.2, and make up to 1000 mL.

[0117] Dissolve the above culture medium and dispense it into test tubes and Erlenmeyer flasks, sterilize at 121°C for 20 min, and cool to obtain a common liquid culture medium;

[0118] Step 2: Take 50 μL of Staphylococcus aureus stored in a glycerol tube and inoculate it into a 10 mL sterilized ordinary NB culture medium test tube. Incubate it in a shaker at 37°C and 150 rpm for 12-16 hours to obtain an activated bacterial solution. Take 500 μL of the activated strain and inoculate it again into 100 mL of fresh ordinary NB culture medium. Continue incubating it in a shaker at 37°C and 150 rpm for 8 hours. Dilute the activated Staphylococcus aureus suspension with the suspension culture medium so that the viable count after dilution is 1×10 5 CFU / mL, that is, the bacterial suspension to be tested;

[0119] Step 3: Take a 96-well plate and add the diluted test bacterial suspension to all wells (outer wells, inner circle wells, and center wells). Cover the plate with a lid and seal the outer ring of the plate with sealing tape.

[0120] Step 4: Place the sample plate in a 37°C incubator and use a microplate reader to measure the absorbance of each well at 600 nm at 0 h and 48 h.

[0121] The positions of the holes in the microplate in this comparative example are as follows: Figure 2 As shown, the microporous plate includes peripheral holes, inner circle holes 1, inner circle holes 2, and a central hole.

[0122] Comparative Example 4: Determination of the Minimum Inhibitory Concentration of Nisin against Enterococcus faecalis

[0123] The minimum inhibitory concentration of nisin against Enterococcus faecalis was determined using the commonly used laboratory procedure. The steps are as follows:

[0124] Step 1: Prepare 500 mL of LB liquid medium according to the culture requirements for Enterococcus faecalis. The composition of the medium, based on mass-to-volume ratio, is as follows: 5.0 g (w / v) tryptone, 2.5 g (w / v) yeast extract, and 2.5 g (w / v) sodium chloride. Dissolve in water and adjust the pH to 7.0 ± 0.2. Make up to 500 mL. Heat at 90–100°C until transparent and clear. Aliquot into test tubes and Erlenmeyer flasks. Sterilize at 121°C for 20 min. Cool to obtain LB medium.

[0125] Step 2: Weigh 0.2000 g of Nisin (nisin) powder and dissolve it in 100 mL of sterile triple-distilled water to prepare a 2000 μg / mL Nisin stock solution. Sterilize the solution by filtration through a sterile 0.22 μm fiber filter in a laminar flow hood, aliquot, and store at -20°C. Thaw the solution before experiment and dilute it to 400 μg / mL in LB medium.

[0126] Step 3: Take 50 μL of E. faecalis stored in the glycerol tube and inoculate it into a 10 mL sterilized ordinary LB medium test tube, and culture it in a shaker at 37°C and 180 rpm for 6-8 h to obtain an activated bacterial solution; take 500 μL of the activated strain and inoculate it again into 100 mL of fresh ordinary LB medium, and expand the culture under the conditions of a shaker at 37°C and 180 rpm; dilute the activated E. faecalis suspension with the suspension medium so that the number of viable bacteria after dilution is 1×10 7 CFU / mL, which is the indicator bacteria suspension to be tested;

[0127] Step 4: Take a 96-well plate and add 200 μL of sterile phosphoric acid solution (0.02 mol / L, pH 7.0) to the outermost circle (outer wells). Add 100 μL of LB medium to the other wells (inner circle well 1, inner circle well 2 and center well); F2 to F11 are set as bacterial growth control groups, G2 to G11 are set as negative control groups, and the remaining wells are experimental groups; the well distribution of each group in this comparative example is as follows Figure 3 As shown;

[0128] Step 5: Add 100 μL of 400 μg / mL Nisin from Step 2 to well B2 and perform gradient dilution using a two-fold gradient dilution method (i.e., each dilution step reduces the concentration of the solution in the previous step to 1 / 2 of the original concentration, thus forming a series of geometric dilution concentration gradients). Perform gradient dilutions with suspension culture medium. The Nisin concentrations in wells B2 to B11 are shown in Table 6. Perform four replicates for each sample, i.e., C2 to C11, D2 to D11, and E2 to E11. The procedures are the same as for wells B2 to B11.

[0129] Step 6: Add 100 μL of Enterococcus faecalis suspension to the bacterial growth control group and the experimental group; add 100 μL of sterile LB medium to the negative control group; and apply sealing tape.

[0130] Step 7: Place the sample plate in a 37°C incubator. Using the OD value method, read and record the OD600 value of each well in the 96-well plate before and after 24 hours of incubation using a microplate reader. Calculate the difference in OD600 values ​​before and after drug exposure. The drug concentration in the wells with an OD value change of less than 0.05 is the minimum inhibitory concentration (MIC).

[0131] The pore distribution of each group in this comparative example is as follows Figure 3 As shown, the glucose oxidase phosphate solution is added last to the outermost circle of wells in the microplate. The outermost circle of wells is marked as glucose oxidase solution wells. The wells in the microplate are also divided into test wells, negative control wells, and bacterial growth control wells.

Claims

1. A method for cultivating facultative anaerobic microorganisms based on a microplate, characterized in that: The following steps are involved: 1) mixing gellan gum with a culture medium for facultative anaerobic microorganisms and melting them to obtain a suspension culture medium; diluting the expanded cultured facultative anaerobic microorganisms with the suspension culture medium to obtain an indicator bacteria suspension; the gellan gum content in the suspension culture medium is 0.2 g / L to 0.4 g / L; 2) Add a phosphate solution containing glucose to the outermost wells of the microplate, add an indicator bacteria suspension to the inner wells of the microplate, then add a glucose oxidase solution to the outermost wells of the microplate, and seal the microplate. 3) static culture; The glucose concentration in the glucose-containing phosphate solution in step 2) is 50 g / L to 300 g / L; the concentration of the glucose oxidase solution is 0.5 g / L to 50 g / L; and in each outermost circle of culture wells, the volume ratio of the glucose-containing phosphate solution to the glucose oxidase solution is 50-100:50-100.

2. The microplate-based facultative anaerobic microbial culture method according to claim 1, characterized in that: The gellan gum in step 1) is deacylated gellan gum or low-acyl gellan gum; The facultative anaerobic microorganism is Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, Enterococcus faecalis, Streptococcus thermophilus, Listeria, yeast or Candida; In each outermost circle of culture wells, the volume ratio of the glucose-containing phosphate solution to the glucose oxidase solution is 1:

1.

3. The microplate-based facultative anaerobic microbial culture method according to claim 1, characterized in that: The viable count of the indicator bacteria suspension in step 1) is 10 4 ~10 7 CFU / mL; that is, the expanded culture of facultative anaerobic microorganisms is diluted with the suspension culture medium so that the viable count after dilution is 10 4 ~10 7 CFU / mL; The glucose-containing phosphate solution in step 2) refers to glucose dissolved in 0.02 mol / L to 0.05 mol / L phosphate buffer solution with a pH of 4.0 to 7.0; The glucose oxidase solution in step 2) refers to glucose oxidase dissolved in 0.02 mol / L~0.05 mol / L, pH 5.0~7.0 phosphate buffer.

4. The microplate-based facultative anaerobic microbial culture method according to claim 1, characterized in that: The amount of the glucose-containing phosphoric acid solution added to the culture well is 50-100 μL, and the amount of the indicator bacteria suspension added to the culture well is 50-150 μL; Step 2) Add 50-150 μL of suspension culture medium to the culture wells; The static culture conditions in step 3) are to culture in an incubator at 28-45°C for 4-72 hours; The melting temperature in step 1) is 90-100°C.

5. The microplate-based facultative anaerobic microbial culture method according to claim 1, characterized in that: The suspension culture medium in step 1) specifically refers to mixing gellan gum with a culture medium for facultative anaerobic microorganisms, stirring and heating at 90-100° C. to melt until transparent and clear, sterilizing, and cooling; The expanded culture of facultative anaerobic microorganisms refers to inoculating the strains of facultative anaerobic microorganisms into a sterilized culture medium, culturing at 28-45°C for 6-16 hours to obtain an activated bacterial solution; taking the activated bacterial solution and inoculating it again into a fresh culture medium, and expanding the culture at 28-45°C; The expanded culture is static culture or shaking culture for 8 to 24 hours.

6. The method according to any one of claims 1 to 5 is used for detecting the activity of antibacterial substances in facultative anaerobic bacteria.

7. A method for detecting the activity of an antibacterial substance of facultative anaerobic bacteria, characterized in that: The following steps are involved: S1. Mixing gellan gum and a culture medium of facultative anaerobic microorganisms, and melting them to obtain a suspension culture medium; S2. diluting the expanded cultured facultative anaerobic microorganisms with the suspension culture medium to obtain an indicator bacteria suspension; S3, after the antibacterial substance is prepared into a mother solution, it is diluted with a suspension culture medium to obtain an antibacterial substance suspension; S4. Divide the inner wells of the microplate except the outermost circle of wells into a test area, a bacterial liquid growth control area, and a negative control area; add a phosphate solution containing glucose to the outermost circle of wells of the microplate, add a suspension culture medium to the other inner wells, then add an antibacterial substance suspension to the wells in the test area, then add an indicator bacteria suspension to the wells in the test area and the wells in the bacterial liquid growth control area, add a suspension culture medium to the wells in the negative control area, and finally add a glucose oxidase solution to the outermost circle of culture wells, and seal the microplate; S5, static culture; S6. Measure the absorbance of each well at 550-600 nm; In step S4, the antibacterial substance suspension is diluted with the suspension culture medium to form different concentrations in the wells of the test area; the different concentrations refer to the antibacterial substance suspension in the wells of the test area being graded diluted with the suspension culture medium so that a concentration gradient of the antibacterial substance is formed in the wells; The glucose concentration in the glucose-containing phosphate solution in step S4 is 50 g / L to 300 g / L; the concentration of the glucose oxidase solution is 0.5 g / L to 50 g / L; in each outermost circle well, the volume ratio of the glucose-containing phosphate solution to the glucose oxidase solution is 50-100:50-100; The concentration of the antibacterial substance suspension in step S3 is 200-1000 μg / mL; The content of the gellan gum in the suspension culture medium in step S1 is 0.2 g / L~0.4 g / L.

8. The method for detecting the activity of antibacterial substances of facultative anaerobic bacteria according to claim 7, characterized in that: In step S4, the gradient dilution is specifically performed using a two-fold gradient dilution method.

9. The method for detecting the activity of antibacterial substances of facultative anaerobic bacteria according to claim 7, characterized in that: The wells of the test area are divided into a first area and a second area. The suspension of the antibacterial substance in the wells of the first area is diluted with a suspension culture medium in a gradient manner so that the antibacterial substance forms a concentration gradient. The wells of the second area are parallel wells for parallel testing. Each well in the first area corresponds to a concentration, and at least one parallel well is provided in the second area for each concentration. The concentration gradient is 0.1 to 400 μg / mL. In step S6, a curve is drawn based on the absorbance value, with the concentration of the antibacterial substance as the horizontal axis and the OD600 value as the vertical axis, and the inflection point is the minimum inhibitory concentration; The viable count of the indicator bacteria suspension in step S2 is 10 4 ~10 7 CFU / mL; The glucose-containing phosphate solution in step S4 refers to glucose dissolved in 0.02 mol / L to 0.05 mol / L, pH 4.0 to 7.0 phosphate buffer; The glucose oxidase solution in step S4 refers to glucose oxidase dissolved in 0.02 mol / L to 0.05 mol / L, pH 5.0 to 7.0 phosphate buffer; The amount of the glucose-containing phosphoric acid solution added to the culture well is 50-100 μL, and the amount of the indicator bacteria suspension added to the culture well is 50-150 μL; The static culture condition in step S5 is to place the culture in an incubator at 28-45°C for 4-72 hours; The melting temperature in step S1 is 90-100°C; The gellan gum described in step S1 is deacylated gellan gum or low acyl gellan gum, The facultative anaerobic microorganism is Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, Enterococcus faecalis, Streptococcus thermophilus, Listeria, yeast or Candida.

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